Chapter 16Cattle Pheromones

16.1. ABSTRACT

Chemical signals play a major role in mammalian reproduction and behavior. While the existence of sexual pheromones and their significance in reproductive behavior are very well known in laboratory mammals, there is little information about the species-specific chemosignal in cattle. However, in the last decade there have been some important and relevant reports for cattle pheromones. This chapter provides a comprehensive account on cattle pheromones and their role in reproduction and social behaviors. Sexual attraction, mother-young interactions, estrus indication, estrus induction, puberty acceleration, reducing the postpartum anestrus, hormonal stimulation, and enhancing the penis erection are some of the classical events influenced by pheromones. During the reproductive cycle of cattle, the male exhibits a specific behavior of flehmen that is used as an indicator to detect estrus. Furthermore, the sensory system involved in the perception of pheromones is mediated through the vomeronasal organ (VNO) and main olfactory system (MOS). The sex attractant volatile compounds have been identified in the urine of the cow, buffalo, and horse. The urinary volatile compounds identified in female buffalo are capable of enhancing the sperm quantity, which can be considered as a significant finding for cattle pheromones. The pig appeasing pheromone (PAP) is characterized from the mother skin used to reduce the fighting behavior among the piglets whereas the boar salivary pheromone is found to induce puberty attainment and boar mate. Similarly, PAP is also present in the horse, cat, dog, and human and plays a significant role. In the goat and sheep, the volatiles identified in the ram and buck wool fleece to stimulate LH and ovulation induction. There is plenty of scope for the application of cattle pheromones in the animal reproduction and management.

16.2. INTRODUCTION

The pheromone signals are known to have a potential role in animal reproduction and management (Archunan 2009; Buck 2000; Dominic 1991; Rekwot et al. 2001; Tirindelli et al. 2009). The sources of chemosignals are urine, feces, vaginal secretions, saliva, and specialized scent glands including the odor produced from hair and wool (Albone 1984; Aron 1979; Patra et al. 2012; Van den Hurk 2007). Communication of the timing of the physiological event of ovulation and coordination of sexual behavior are important for successful fertilization (Schams et al. 1977; Ziegler et al. 1993); the success rate of artificial insemination in cattle mainly depends on the time of estrus it is inseminated. If the female fails to conceive when it is inseminated in the nonestrus period, it is a great economic loss estimated around $300 million to the dairy industry in the United States and in India the approximate loss would be several crores. The detection of estrus and diagnosing early pregnancy are the major problems in farm animals, particularly in buffalo. The other problems like irregular or prolonging of the estrous cycle, anestrus, fighting behavior among young ones, mother-young bond, unmotivated males, and poor farm animal management including stress are considered as major issues in farm animals (Rekwot et al. 2001) that need to be addressed. Moreover, pheromones have not been exploited for the purpose of enhancing livestock production.

The animal releases volatile odors into the surrounding atmosphere, most of which are waste products of metabolism in which emission of some compounds closely related to reproductive activities are termed as chemical signals (Hradecky 1975). The estrus-related odors are present only during the preestrus and estrus stages. These chemical signals have been reported to be volatile and nonvolatile molecules that are perceived through the main or accessory olfactory system (Brennan and Keverne 1997; Tirindelli et al. 1998). In mammals, structurally and anatomically, the olfactory systems are classified into two types: the MOS and the accessory olfactory system (AOS), specialized for the detection and transmission of pheromonal information (Halpern and Marinez-Marcos 2003; Mucignat-Caretta et al. 2012).

In the last two decades, there has been a considerable increase in the knowledge of the chemistry of pheromones in cattle (Rajanarayanan and Archunan 2011; Rameshkumar et al. 2000; Sankar and Archunan 2004; Sankar et al. 2007), pig (Signoret 1970), horse (Kimura 2001), goat (Delgadillo et al. 2006), and sheep (Gelez and Fabre-Nys 2006). These findings suggest that cattle pheromones may act together for influencing precopulatory behavior and successful reproduction. It further indicates that cattle pheromones may be a single compound or a mixture of compounds and that each of the major fractions was faithfully involved in conveying specific signals related to reproductive and social behaviors. The pheromones have not been exploited as much as they can for farmer utility although they have a tremendous role in reproduction. The present review provides insights about the nature of the compounds, behavioral characterization, and practical utility of cattle pheromones that are available (Table 16.1).

16.3. BIOSTIMULATION

Pheromones in the urine, feces, or from cutaneous glands can be perceived through the olfactory system to elicit both behavioral and endocrine responses in conspecifics, and biostimulation (priming pheromones) can exert profound effects on reproductive activity via the hypothalamic system (Dulac and Wagner 2006; Rekwot et al. 2001). The role of pheromones has been shown in several reproductive events such as sexual maturity, induction of ovulation, reduction of postpartum anestrus, and coitus in many mammalian species including rodents, wild animals, feral populations, swine, sheep, goat, and cattle (Burns and Spitzer 1992).

The exposure to boars induced puberty at an earlier age than gilts reared without being exposed to a boar and reduced the postpartum period in lactating sows indicates the potential role of primer pheromone (Brooks and Cole 1970; Kirkwood et al. 1981). The “ram effect” (primer pheromone) has been reported to accelerate the onset of estrus activity and promote varying degrees of estrus synchronization (Schinckel 1954). It is important to note that the ewes of most breeds are anestrus for some portion of the year and exposure of ram to anestrus ewes reduces the length of the anestrus period leading to the return of estrus activity (Rekwot et al. 2001).

Biostimulation is an inexpensive and suitable method for extensive management systems and it is an excellent example of the potential underlying “control systems technologies aimed at controlling reproductive performance” (Martin 1995; Signoret 1970). The role of primer pheromones in cattle reproduction is not as clearly defined as that in other species such as sheep, goats, and swine, possibly due to nutritional and other environmental stresses (Roberson et al. 1991). It is important to note that the presence of a teaser bull did not hasten puberty or alter the size of ovaries in a group of prepubertal heifers (Berardinelli et al. 1978; Macmillan et al. 1979; Roberson et al. 1987).

One of the main problems in cattle reproduction is anestrus, defined as absence of the estrous cycle, which has to be decreased in order to obtain a more sustainable production in the livestock industries (Fiol and Ungerfield 2012). Biostimulation (male or bull effect) can be defined as the stimulus provoked by the presence of males, which induces estrus and ovulation through genital stimulation, pheromones, or other external cues (Chenoweth 1983). The stimulus provoked by the introduction of the males can act through different pathways, including olfactory, visual, and auditory signals (Ungerfield 2007). In cattle, males’ excretory products and cervical mucus from estrus females enhance ovarian function, both in postpartum cows (Berardinelli and Joshi 2005; Wright et al. 1992) and prepubertal heifers (Izard and Vandenbergh 1982).

Berardinelli and Joshi (2005) evaluated resumption of cyclic activity in postpartum, anovular, primiparous cows exposed to bulls or the excretory products of bulls. The cows were allowed in the pens along with a male for 12 hours daily during 70 days of experiments. The authors found that anestrus postpartum length was greatly reduced when the female was exposed to males or the excretory products of males, suggesting that the biostimulatory role of bulls appears to be mediated by pheromone present in their excretory product. The nature of the compound present in the excretory product is not yet identified (Berardinelli and Tauck 2007; Fernandez et al. 1993). Burns and Spitzer (1992) observed a similar reduction in postpartum interval to first estrus in cows exposed to bulls or to androgenized females. The effects of male exposure on cyclic activity have been evaluated mainly in Bos taurus taurus and other possible effects of biostimulation in Zebu (Bos taurus indicus), both in the postpartum cow (Rekwot et al. 2001; Soto Belloso et al. 1997), and prepubertal heifer (Rekwot et al. 2001). The biostimulation was also found effective to reduce the anestrus period in female buffaloes, Bubalus bubalis (Gokuldas et al. 2010; Ingawale and Dhobe 2004).

Improving reproductive efficiency in beef and dairy cattle should be one of the main objectives to obtain a more sustainable production. In this context, sociosexual stimulus, like biostimulation, represents low-cost and hormone-free alternatives when used alone or in conjunction with other techniques to increase reproductive results.

16.4. COW PHEROMONES: SOURCES AND IDENTIFICATION OF CHEMOSIGNALS

In ungulates, bulls routinely investigate the urine of anogenital areas of females presumably to determine their reproductive state. The involvement of chemosignals in the cow’s reproductive process is well documented; the female produces a specific odor during estrus in urine that is perceived by the bull (Archunan and Rameshkumar 2012; Denhard and Claus 1996; Rameshkumar et al. 2000). The dispersion of estrus-specific compounds in the cow’s body fluids has been demonstrated previously in swabs from the fluids (vaginal secretion, urine, milk, and blood) (Kiddy and Mitchell 1984; Rivard and Klemm 1989). The presence of estrus-specific volatiles was confirmed in milk, but the study did not find any compounds that were qualitatively different between stages but found there were significant quantitative differences in 15 compounds in milk (Weidong et al. 1997).

Klemm et al. (1987) found that blood acetaldehyde levels decreased rapidly just before, or at the onset of estrus, and suggested that estrus and ovulation could potentially be predicted by monitoring the levels of acetaldehyde in milk, saliva, sweat, or breath. Acetaldehyde was also found to be estrus-specific in bovine vaginal secretions (Weidong et al. 1997); however, it has been tested in a bull bioassay previously (Presicce et al. 1993) and was not behaviorally active when tested as a singular component.

Several studies have focused on vaginal secretions found during estrus. Preti (1984) patented a method for detecting bovine estrus based on the quantification of methyl heptanol in vaginal secretion. Another finding showed that the concentration of free fatty acids in estrus vaginal discharge increased gradually before estrus and decreased rapidly thereafter (Hradecky 1986). By contrast, the concentration of free fatty acids in urine, but not in vaginal discharge, was affected by the luminal concentration. Klemm et al. (1987) found nine estrus-specific compounds in urine that were tested positively in a bull behavioral assay. These compounds included two ketones, four amines, one alcohol, one diol, and one ether. Sankar and Archunan (2004) reported that other body fluids such as vaginal fluid, saliva, feces, and milk collected from estrus period contained chemical cues that attract the opposite sex.

Bulls can discriminate estrus from nonestrus urine and estrus urine has been shown to elicit sexual behavior in the cow. Rameshkumar et al. (2000) identified two estrus-specific compounds (i.e., 1-iodo undecane and di-n-propyl phthalate), however, the behavioral assay later clearly indicated that the 1-iodo undecane is capable of stimulating the bull response for coitus and hence was considered as a biochemical marker for bovine (Archunan and Rameshkumar 2012).

Sankar and Archunan (2004) studied the behavioral assay among the proestrus, estrus, and diestrus samples; the test clearly showed that the estrus vaginal fluid was found to be more variable (p < 0.001) than the saliva, feces, and milk. The vaginal secretions induced the maximum response in bulls showing flehmen behavior, thereby confirming that the vaginal secretions from the estrus cow contained pheromone(s), which along with urinary pheromone(s) result in attracting the bull.

Furthermore, our laboratory study reported that 11 different volatiles are identified in cow saliva from three different reproductive phases (Sankar et al. 2007). Among the identified compounds, the following compounds, trimethyl amine, acetic acid, phenol 4-propyl, pentonic acid, and propionic acid were specific to estrus. The behavioral assay has confirmed that the compound from a salivary source (i.e, trimethylamine) may be involved in attracting the males. Interestingly, the chemical profiles of estrus feces differed significantly from other phases by specific substance such as acetic acid, propionic acid, and 1-iodo undecane (Sankar and Archunan 2008); the behavioral assay also confirmed that these three compounds elicit a series of reproductive behaviors in the bull. It is evident that LH pulse frequency was greatly enhanced in the heifer by exposure to vaginal mucus and urine collected from estrus cows (Nordeus et al. 2012); however, the chemicals responsible for influencing LH alteration are not known. The above investigation on cow pheromone and behavioral characterization have convincingly showed that urine, feces, vaginal secretion, saliva, and milk appear to be prominent sources for cow pheromone production for estrus indication and influence several reproductive behaviors including hormonal stimulation.

Another interesting aspect is to detect estrus by interspecific communication. The ability of dogs (Hawk et al. 1984), rats (Denhard and Claus 1988), and mice (Rameshkumar et al. 2008; Sankar and Archunan 2005) to detect estrus-specific odor in cow urine is reported. Trained dogs can distinguish the cow luteal phase (Hawk et al. 1984; Kiddy and Mitchell 1984). In addition, a detailed experimental study was carried out using the mouse as an experimental model to evaluate which of the body fluids of the estrus cow has more attracting ability towards mice (Sankar and Archunan 2004). The test clearly showed that the estrus vaginal fluid was found to be the most attractive source followed by saliva, feces, and milk. The findings suggest that the specific odors present in the estrus phase not only attract the conspecific but also attract between species.

16.5. BUFFALO PHEROMONES

The occurrence of silent ovulation without any behavioral symptoms of estrus is the major problem in buffalo reproduction (Danell et al. 1984; Dobson and Kamonpatana 1986). Unlike in the cow and other ungulates, visual signs of estrus are not prominent in the buffalo, which make it difficult to detect heat effectively. Hence, there is a need for a reliable method to detect estrus in the buffalo. Recent reports show that the buffalo exhibits a reproductive behavior and releases chemosignals from few sources that can be exploited for estrus detection (Archunan 2009). Rajanarayanan and Archunan (2004) demonstrated that the average number of all flehmen behavior (2.03 ± 0.66) and repeated flehmen (1.05 ± 0.64) behavior in the bull upon exposure to an estrus heifer were significantly higher than those of diestrus periods of all (0.69 ± 0.25) and repeated flehmen (0.11 ± 0.10).

The nature of the volatiles present in the urine is confirmed and proved to be a pheromonal signal (Rajanarayanan and Archunan 2011). Fourteen different volatile compounds from all the stages of the estrous cycle were identified in urine samples, which included phenol, ketone, alkane, alcohol, amide, acid, and aldehyde. Amongst the identified compounds, a few, such as 2-octanone, 2-methyl-N-phenyl-2 propenamide, decanoic acid, N,N-bis (2 hydroxy ethyl) dodecanamide, tetradecanoic acid, and hexadecanoic acid, were commonly found throughout the cycle (preestrus, estrus, postestrus, and diestrus). However, the volatile compounds 1-chlorooctane, 4-methyl phenol, and 9-octadecenoic acid occurred only during estrus. Behavioral assay further revealed that bulls were attracted and exhibited repeated flehmen behavior toward 4-methyl phenol, whereas bulls exhibited penis erection and mounting response by exposure to 9-octadecenoic acid. By contrast, the other compound 1-chlorooctane did not show any such sexual behavior in bulls. It is interesting to note that these compounds are absent during postestrus and diestrus, which indicate that these compounds are characteristic to the estrus period. The reports convincingly conclude that 4-methyl phenol acts as a sex attractant compound and 9-octadecenoic acid acts as mounting response. It is remarkable to note that the urinary sex pheromones have been shown to enhance the sperm production after exposing to the nasal region; this has been awarded a patent (Archunan and Rajanarayanan 2010).

The nature of the volatile compounds produced during estrus in buffalo has been predicted. The existence of compounds phenol, 3-propyl phenol, and 9-octadecenal in preestrus urine suggests that these three volatiles may be considered as the preindicators for estrus. It is also interesting to note that the volatile phenol present in preestrus probably becomes 4-methyl phenol in estrus urine by the addition of a methyl group. Similarly, 9-octadecenoic acid, which appears in estrus, is probably derived from the preestrus compound of 9-octadecenal. Presumably, a change occurs in the formation of chemical compounds from the preestrus to estrus phase.

In addition to sex pheromone characterization in urine, other body fluids such as feces, vaginal mucus, and saliva have evinced great interest in view of identification of the volatile compounds and their behavioral assay, which may be considered as another important source for sex pheromone production. A specific volatile compound, 9-octadecenoic acid, has been identified as estrus-specific in vaginal mucus (Karthikeyan and Archunan 2013); it is to be remembered that the volatile compound 9-octadecenoic acid is already identified in estrus urine of the buffalo (Rajanarayanan and Archunan 2011). In feces, two volatile compounds, 4-methyl phenol and trans-verbenol, have been identified and confirmed as estrus indicator, suggesting that feces may be considered as a source of estrus indicator in the buffalo (Karthikeyan et al. 2013). The 4-methyl phenol (P-cresol), a unique volatile compound, can be considered as a common compound of estrus-specific since it is present in urine (Rajanarayanan and Archunan 2011), feces (Karthikeyan et al. 2013), and saliva (Karthikeyan 2011). Hence, this compound might be produced under the influence of estrus hormones.

16.6. BOAR PHEROMONES

As far as the boar pheromone is concerned, three types of pheromones have been recognized (1) boar mate pheromone, (2) puberty accelerating pheromone, and (3) appeasing pheromone. Chemical communication in the pig is notably mediated by saliva. The boar mate pheromone produced from pig saliva against the estrus sow is remarkably involved in the organization of the sequence of sexual behavior to attract the estrus females and facilitate the display of a receptive posture (Signoret 1970). The steroid pheromones such as 5α-androsterone and 3α-androsterone induce the appropriate standing response for mating and these odors are essential for a prolonged act of coitus in pigs. An aerosol boar mate is now marketed to aid pig artificial insemination practice by inducing the immobilization reflex in the estrus female (Booth 1984). The puberty accelerating pheromone is also released from saliva in the male pig, which is reported to induce early puberty in young female piglets (Pageat and Teyssier 1998). The boar pheromones have been shown to accelerate puberty in gilts by about 30 days to synchronize estrus and to reduce the postpartum period in lactating sows (Brooks and Cole 1970). Furthermore, those gilts reached early puberty by the presence of a boar odor, have higher ovulation rates, regular estrous cycles, and improved reproductive potential than the controls (Izard 1983).

Appeasing pheromone is another important chemical communication involved between mother and young pups of pigs. Generally, after weaning, the piglets will fight when group-housed, which leads to reduce greatly the feeding among piglets. The fighting behavior is a major concern among the farmers because the pigs do not gain weight due to underfeeding. Odors isolated from the skin of milking sows have been shown to reduce agonistic behavior in piglets (Pageat and Teyssier 1998). The putative maternal pheromone is composed of six fatty acids in different proportions (Pageat 2001): hexadecanoic acid, cis-9-octa decenic acid, 9,12-octyladecanoic acid, dodecanoic acid, tetradecanoic acid, and decanoic acid. The commercial synthetic analog had similar effects when tested in industrial husbandry (McGlone and Anderson 2002). This pheromone can be considered as an outstanding discovery in the mammalian pheromones.

16.7. SMALL RUMINANT PHEROMONES: GOAT AND SHEEP

The small ruminants such as the goat and sheep are economically important farm animals worldwide for their milk, meat, and wool. The response of the female goat to the male is weak but can be induced by the introduction of teaser bucks that had previously been exposed to long days (Delgadillo et al. 2006; Rivas-Munoz et al. 2007). The odor of the male and its sexual behavior plays a primary role in inducing ovulation, while vocalizations appear to facilitate the display of the does’ estrus (Delgadillo et al. 2006). Additionally, the sebaceous gland has been indicated as sources of primer pheromone production in goats (Iwata et al. 2000; Sugiyama et al. 1981). The primer pheromones produced by the male have been known to stimulate the reproductive neuroendocrine system of anestrus animals (Chemineau 1987; Knight and Lynch 1980). Exposure of a male into a group of anestrus females leads to the activation of sexual activity by influencing the LH secretion and synchronization of ovulation in the sheep and goat (Knight et al. 1978; Ungerfeld et al. 2004). Just as the influence of primer pheromones in the acceleration of puberty in rodents is well established, the chemosginals from the ram and buck have been found to accelerate puberty in the sheep and goat (Shelton 1978; Underwood et al. 1944). The evidence indicates that 4-ethyl octanoic acid (4EOA) identified in major fleece does not evoke an LH response in a female conspecific but its derivatives might have pheromonal activity (Iwata et al. 2003). It was also found that the synthetic 4EOA possessed a releaser pheromone activity that induced specific behavior in the recipient, and female goats showed some interest in the odor (Iwata et al. 2003; Sasada et al. 1983).

The pheromones secreted through the ewe’s wool and wax (Tilbrook and Cameron 1989) and vaginal secretions (Ungerfeld and Silva 2004) sexually attract the ram; however, the nature of the pheromonal compound present in the female is not yet investigated. The volatile substance(s) responsible for female LH secretion has been found in male sheep hair, skin, and their extracts obtained by organic solvents (Cohen-Tannoudji et al. 1994). It is further reported that the compounds 1, 2-hexadecanediol and 1, 2-octadecanediol appear to be responsible for this pheromonal effect in sheep. The synthetic compounds are reported to be effective in stimulating LH release in anestrus ewes. In the domestic sheep, primer pheromones are considered the most important signals involved in sociosexual stimulation of the reproductive processes and male-female interactions induce changes in the pulsatile rhythm of the LH secretion in both sexes, which influences reproductive endocrinology.

The occurrence of olfactory memory for the formation of mother-young bond between ewes and lambs is another interesting aspect of a milestone in support of the involvement of pheromonal communication. Sheep have been used as the best model to study the mother-young relationship (Bouissou 1968; Kendrick et al. 1992; Nowak et al. 2011). The strong bond between ewe and lamb formed shortly after parturition particularly within few hours is very crucial and critical for lamb survival (Keller et al. 2005; Porter et al. 1991). The onset of maternal responsiveness and the development of mother-young attachment in sheep are under the control of hormonal and sensory stimulation (Kendrick et al. 1992; Keverne et al. 1993; Nowak et al. 2011). Offspring recognition through olfaction in most females is generally believed to be based on the offsprings’ individual olfactory signature (Poindron et al. 1988; Porter et al. 1991), which is believed to originate from its body coat (Alexander and Stevens 1982) from the anal region (Alexander et al. 1983). It is reported that majority of the odors from the anal region in animals have been associated with pheromones (Bean 1982). The first day of life is entirely dependent on the success of the first suckling episodes (Nowak et al. 2011). If suckling is prevented during the first few hours after birth by covering the ewes udder, the development of a preference for mother is lowered (Nowak et al. 1997), suggesting that sensory outputs from emanating the suckling influence the development of filial attachment.

Amniotic fluid is an important substance in sheep for the establishment of maternal responsiveness toward neonates, facilitating their initial acceptance by the mother (Poindron et al. 2010). Amniotic fluid also carries individual olfactory cues from the neonates that help the formation of the maternal bond. Recently, several volatile organic compounds from lamb wool have been identified; these compounds serve as olfactory cues for neonatal recognition (Burger et al. 2011). However, the exact chemosignals involved in the maternal responsiveness toward the lamb are yet to be determined. It is known that the cell proliferation and survival in the adult brain are influenced by several internal and external factors (Lledo et al. 2006); for instance, the physiological status of pregnancy and parturition has been found to regulate cell proliferation in rodents. Regarding the establishment of maternal selectivity, it is hypothesized that downregulation of cell proliferation occurs in specific areas of the sheep brain and as well as in the main olfactory bulb during the early postpartum period (Brus et al. 2010; Levy et al. 1990). This could facilitate the development of olfactory perpetual memory that is retained in favor of the survival of the newborn neurons that might somehow assist in the learning process.

16.8. HORSE PHEROMONES

The role of biostimulation in the horse is very much needed in the context of horse breeding; however, the research on chemical communication in the horse is very much limited. Mares come into estrus several times a year (i.e., horses are a seasonal polyestrus species). A mare is sexually receptive towards a stallion for 5–7 days and ovulation occurs in the final 24–48 hours of estrus (Kiley-Worthington 1987). The precise determination of the estrus period is difficult and special qualification is needed to effectively elucidate the proper time of insemination (Mozuraitis et al. 2012). A mare signals estrus by urinating in the presence of a stallion, raising her tail, and revealing the vulva. A stallion approaching with a high head will usually nudge and nip a mare, as well as sniff her urine to determine her readiness for mating (Kiley-Worthington 1987).

Ma and Klemm (1997) detected 45 urinary volatile compounds at the estrus and diestrus stage of mares. Mozuraitis et al. (2012) analyzed the estrus urine samples and found 150 urinary volatiles; among the compounds, m- and p-cresols occurred significantly in greater amounts in estrus when compared to nonestrus. A great increase in amounts of p-cresol in urine samples from all mares of different breeds during the most active stallion acceptance periods provides a good signal to stallion that a mare is in estrus; thus p-cresol might be consider as a sex pheromone component. It is further reported that p-cresol is able to influence the penile erection in horse (Buda et al. 2012). In addition to urinary sources, sex pheromones such as palmitic and miristic acid are identified in feces and found to show the estrus signal to attract a stallion (Kimura 2001). The finding indicates that sex pheromones are produced from more than one source as it is already recorded in the cow and buffalo.

16.9. ODORANT BINDING PROTEINS IN CATTLE

Odorant binding proteins (OBPs) are the soluble proteins of the lipocalin superfamily found in the mucus layer lining the olfactory epithelium of mammals (Mitchell et al. 2011; Pelosi 1994). The OBP is almost of similar structural sequence to the pheromone carrier protein that is present in almost all the sources of pheromone production in mammals (urine, saliva, sweat, vaginal mucus, etc.), and the prime function of the pheromone carrier protein is to bind the pheromone compounds and release them to the environment for manifesting the effect. OBPs are the first in the relay mechanism in pheromonal chemical reception because they provide the link between the chemical signal present in the environment and the odorant receptors located in the olfactory and peripheral sensory systems in mammals (Pelosi 1994). Major functions have been suggested for OBPs, such as scavenging odorants (Vincent et al. 2004) and protecting the airways (Mitchell et al. 2011). The OBP is well documented in bovine mucosa (Pelosi 1994) and the 1-octen-3-ol has been found to be the natural ligand of bovine OBP. Since 1-octen-3-ol is an important component of bovine breath and a very potent attractant for many insects, this indicates that the role of bovine OBP may be to clear the breath of 1-octen-3-ol to reduce the attraction of insects (Ramoni et al. 2002).

In the buffalo, OBP is reported in saliva; this OBP undergoes posttranslational modification (Rajkumar et al. 2010) and it is presumed that it may bind with the salivary pheromone molecule 4-methyl phenol. In addition, OBP has been found in the nasal mucus of the buffalo, suggesting that OBP may bind the odorants for further processing. In the pig, the nasal mucosal OBP has been reported and it has a specific role to bind with the steroid pheromone molecule (Scaloni et al. 2001). Available evidence on OBP in vertebrates concludes that OBP plays a pivotal role in the perception of the odorant/pheromones, contributing to all process. Furthermore, the previous investigation suggests that the binding and metabolic activity of the urine odorant 5α-androstan-3-one involved the odorant binding process in sheep olfactory mucosa (Krishna et al. 1995).

16.10. APPLICATION OF PHEROMONES IN CATTLE

The potential role of pheromones has been updated in several aspects so as to enhance cattle reproduction and livestock management. Based on the large number of data obtained so far in cattle pheromone, the following applications are derived:

• 1. Puberty acceleration by male pheromone. Advancement of puberty is one of the important reproductive events that has been well established in cattle. The male pheromone is capable to advance the puberty in females than the attainment of normal puberty in several species of mammals including sheep, goat, pig, and cow (Rekwot et al. 2001). This enhancement of puberty in young cattle is considered to have a potential role in cattle production.
• 2. Estrus synchronization and estrus induction in anestrus by male pheromone. Introduction of a male into a group of anestrus females during the nonbreeding season results in the activation of LH secretion and synchronization of ovulation in the sheep and goat. This event is generally called the male effect and is widely used in husbandry of these species. The sources of the male pheromones are reported to be produced in fleece (van den Hurk 2007).
• 3. Postpartum anoestrus reduction by male pheromone. Prolonged postpartum anestrus in primiparous cattle is a major cause for failing to rebreed or breeding late in the breeding season (Short et al. 1990). This prolonged postpartum event results in huge economic loss for farmers. In the cow, this critical problem is easily solved by continuous exposure to bull urine by reducing the postpartum anestrus interval during the first calf suckling (Custer et al. 1990; Fernandez et al. 1996). However, there is little controversy regarding the presence of pheromones in bull urine and the way in which the postpartum anestrus cow could be treated with various protocols (Van den Hurk 2007). Further, the nature of the pheromone signal present in the bull urine is not yet identified.
• 4. Influencing the standing posture by male pheromones. The steroid chemosignals identified in boar saliva have been demonstrated to evoke the immobilization reflex to sows in heat and characterize their readiness to mate (Brooks and Cole 1970). The boar steroid pheromone is available as a boar-mate to pig farmers to assist in artificial insemination.
• 5. Estrus indication by female pheromones. Effective estrus detection would greatly help cattle reproduction by artificial insemination. However, the occurrence of silent ovulation without any behavioral sign of estrus is a major problem in buffalo reproduction (Dobson and Kamonpatana 1986). The successful identification of urinary sex pheromones in the buffalo provides a tool to overcome this problem (Rajanrayanan and Archunan 2011) The urinary volatiles characterized in the cow (Archunan and Rameshkumar 2012; Rameshkumar et al. 2000) and horse (Buda et al. 2012) with reference to estrus is another example of estrus-indicating pheromones. An estrus may be detected in the cow by simply evaluating the level of concentration of a specific volatile at least 0.1 µgm/gm from vaginal secretion. The estrus-indicating volatile compound could be present in several sources, such as urine, feces, saliva, and vaginal mucus of the cow and buffalo. The reports consider that the volatiles specifically present during estrus may be used as a marker for estrus identification (Preti 1984). An attempt is being made to develop a kit to detect estrus in buffalo based on the volatile compounds from our research team.
• 6. Penis erection and enhance the sperm quantity by female pheromones. The urinary sex pheromones have been investigated and proved to enhance penis erection and increase the amount of sperm in buffalo considerably (Archunan and Rajanarayanan 2010); an Indian National Patent was obtained for this finding. The mare urinary pheromone during estrus is reported to influence the penis erection in the stallion (Wierzbowski and Hafez 1961), indicating that the female sex pheromones can be used in assisted reproductive technology for special circumstances in certain cattle.
• 7. Reducing the fighting behavior by maternal pheromones. The discovery of maternal (appeasing) pheromones involved in reducing the agonistic behavior in piglets and stimulating their feeding behavior resulting a significant increase in weight gain is a classical example of the practical application of mammalian pheromones. A synthetic analog called porcine appeasing pheromone is available in the market and is very well used by farmers in the pig industry (Suilence^R) (Pageat 2001). As in pigs, a synthetic pheromone product based on natural compound is commercially available as equine appeasing pheromone (EAP) (Pherocalm^R in Europe; Modipher EQ^R in the United States) (Riley et al. 2002). The application of the appeasing pheromone as pheromonetherapy is well documented, which enables a simplification of treatment for anxiety- and phobia-related issues in various species (dogs, cats, and rabbits) (Gaultier et al. 2005; Griffith et al. 2000; McGlone and Anderson 2002).
• 8. Maternal responsiveness by neonatal pheromones. The olfactory recognition in sheep between the postparturient mother and her offspring is immediately established within the first hour after delivery and helps the mother to accept her own young to the udder (Nowak et al. 2011). The establishment of the maternal selectivity mainly relies on the mother learning the olfactory individual signature of her lamb (Levy and Fleming 2006; Porter et al. 1991). The brain areas, particularly the cortical and medial amygdala, are reported to be involved in the formation of olfactory offspring memory in sheep (Keller et al. 2004; Meurisse et al. 2009), suggesting that neurological brain networks actually sustain the lamb memory (Keller et al. 2005). Although peptide hormones such as oxytocin and vasopressin are released in the olfactory bulb, further investigation showed that oxytocin release alone in the olfactory bulb at parturition is reported to facilitate the recognition of lamb odors by modulating noradrenaline (NA), acetylcholine (ACh), and γ-aminobutyric acid (γGABA) release, which is of primary importance for olfactory memory (Levy et al. 1995). However, the nature of the odor produced from the lamb is yet to be detected for the purpose of future applications.

16.11. FUTURE PERSPECTIVES

Based on the data available, cattle pheromones can be used as efficient tools to improve reproduction and management. In order to control the calving interval, optimized milk production, and maximize offspring in dairy cattle, artificial insemination (AI) is widely applied in farm animal reproduction. The right timing of AI can be achieved by accurate detection of estrus. Therefore, it is possible to develop a kit for easy estrus detection in cattle, particularly for buffalo. The estrus kit can also be developed and used for critically endangered animals since estrus-specific urinary volatile compounds have also been identified in the black buck (Archunan and Rajagopal 2013). It is interesting to note that Weigerinck et al. (2011) have now taken the first steps toward the development of a practical eNose product consisting of an array of sensors based on the sex pheromones in cow feces, and it is important and worth encouraging the development of a product such as BOVINOSE for estrus detection purpose. Since a number of estrus-specific volatile compounds have been characterized in cattle it would be possible to design an effective biosensor for estrus detection.

The role of the bull in reducing postpartum anestrus is extremely important. Hence, information on the nature and function of this pheromone needs to be discovered. This is a challenging task and researchers should be encouraged to pay more attention to this aspect. Appeasing pheromones have plenty of applications, therefore it is necessary to extend the usage of this pheromone in several mammalian species. As the urinary pheromone is able to stimulate the pituitary hormone, a detailed study could be made to consider the future applications of the pheromone-hormone relationship with reference to reproduction.

Pheromonetherapy is used in the dog and cat (Levine and Mills 2008), and this can be extended to other mammalian species including cattle since it has great potential to make the animal calm, reduce anxiety and phobia, and increase grooming in all clinical aspects. The neonatal pheromones have been reported to play a crucial role in the establishment of mother-young relationship in the sheep. In this context, it would be of great interest and there would be much practical utility in characterizing the pheromone compounds responsible for the mother-young bond relationship. Recent findings demonstrate that the bitch’s sex pheromone enhances the heart rate of the dog without showing any other sign of arousal (Dzięcioł et al. 2012). Hence, male and female sex pheromones can be analyzed with special reference to health aspects in cattle and humans.

ACKNOWLEDGMENTS

GA thanks his research scholars who contributed significant data on cattle pheromones. We thank Dr. Chellam Balasundaram for critical reading of the chapter. The research work on pheromones is well supported by Bharathidasan University, DST, DBT, ICAR, UGC, UGC-SAP, and DST-PURSE, Government of India.

REFERENCES

• Abbasi M. The vomeronasal organ in buffalo. Italian J. Anim. Sci. 2007;6:2.
• Achiraman S, Ponmanickam P, Sankar Ganesh D, Archunan G. Detection of estrus by male mice: Synergistic role of olfactory-vomeronasal system. Neurosci. Lett. 2010;477:144–148. [PubMed: 20434521]
• Albone E.S. Mammalian Semiochemistry. John Wiley & Sons; New York: 1984.
• Alexander G, Stevens D. Odour cues to maternal recognition of lambs: An investigation of some possible sources. Appl. Anim. Ethol. 1982;10:165–175.
• Alexander G, Stevens D, Bradley L.R. Washing lambs and confinement as aids to fostering. Appl. Anim. Ethol. 1983;10:251–261.
• Archunan G. Vertebrate pheromones and their biological importance. J. Exp. Zool. India. 2009;12:227–239.
• Archunan G, Rajagopal T. Detection of estrus in Indian blackbuck: Behavioural, hormonal and urinary volatiles evaluation. Gen. Comp. Endocrinol. 2013;181:156–166. [PubMed: 23229002]
• Archunan G, Rajanarayanan S. Composition for enhancing bull sex libido. 2010. Indian Patent No. 244991, dated December 28, 2010.
• Archunan G, Rameshkumar K. 1-Iodoundecae an estrus indicating urinary chemosignal in bovine (Bos taurus). J. Vet. Sci. Tech. 2012;3:121–123.
• Aron C. Mechanisms of control of the reproductive function by olfactory stimuli in female mammals. Physiol. Rev. 1979;59:229–284. [PubMed: 108689]
• Balakrishnan M, Alexandar K.M. Sources of body odour and olfactory communication in some Indian mammals. Ind. Rev. Life Sci. 1985;5:277–313.
• Bean N.J. Olfactory and vomeronasal mediation of ultrasonic vocalizations in male mice. Physiol. Behav. 1982;8228:31–37. [PubMed: 7079320]
• Berardinelli J.G, Joshi P.S. Initiation of postpartum luteal function in primiparous restricted-suckled beef cows exposed to a bull or excretory products of bulls or cows. J. Anim. Sci. 2005;83:2495–2500. [PubMed: 16230645]
• Berardinelli J.G, Tauck S.A. Intensity of the biostimulatory effect of bulls on resumption of ovulatory activity in primiparous, suckled, beef cows. Anim. Reprod. Sci. 2007;99:24–33. [PubMed: 16713140]
• Berardinelli J.G, Fogwell R.L, Inskeep E.K. Effect of electrical stimulation or presence of a bull on puberty in beef heifers. Theriogenology. 1978;9:133–138.
• Bland K.P, Jubilan B.M. Correlation of flehmen by male sheep with female behaviour and oestrus. Anim. Behav. 1987;35:735–738.
• Blissitt M.J, Bland K.P, Cottrell D.F. Detection of oestrus-related odour in ewe urine by rams. J. Reprod. Fertil. 1994;101:189–191. [PubMed: 8064680]
• Booth W.D. A note on the significance of boar salivary pheromones to the male-effect on puberty attainment in gilts. Anim. Prod. 1984;39:149–152.
• Booth K.K, Katz L.S. Role of the vomeronasal organ in neonatal offspring recognition in sheep. Biol. Reprod. 2000;63:953–958. [PubMed: 10952943]
• Bouissou M.F. Effet de l’ablation des bulbes olfactifs sur la reconnaissance du jeune au sa mère chez les ovins. Rev. Comp. Anim. 1968;3:77–83.
• Brennan P.A, Keverne E.B. Neural mechanisms of mammalian olfactory learning. Prog. Neurobiol. 1997;51:457–481. [PubMed: 9106902]
• Brooks P.H, Cole D.J. The effect of the presence of boar on the attainment of puberty in gilts. J. Reprod. Fertil. 1970;23:435–440. [PubMed: 5531082]
• Brus M, Meurisse M, Franceschini I, Keller F, Levy F. Evidence for cell proliferation in the sheep brain and its down-regulation by parturition and interactions with the young. Horm. Behav. 2010;58:737–746. [PubMed: 20692260]
• Buck L.B. The molecular architecture of review odor and pheromone sensing in mammals. Cell. 2000;100:611–618. [PubMed: 10761927]
• Būda V, Mozūraitis R, Kutra J, Karlson A.K.B. p-Cresol: A sex pheromone component identified from the estrous urine of mares. J. Chem. Ecol. 2012;38:811–813. [PubMed: 22592336]
• Burger B.V, le Roux M, Marx B, Herert S.A, Amakali K.T. Development of second-generation sample enrichment probe for improved sortive analysis of volatile organic compounds. J. Chrom A. 2011;1218:1567–1575. [PubMed: 21333300]
• Burns P.D, Spitzer J.C. Influence of biostimulation on reproduction in postpartum beef cows. J. Anim. Sci. 1992;70:358–362. [PubMed: 1548196]
• Chemineau P. Possibilities for using bucks to stimulate ovarian and oestrous cycles in anovulatory goats–A review. Livest. Prod. Sci. 1987;17:135–147.
• Chenoweth P.J. Reproductive management procedures in control of breeding. Aust. J. Anim. Prod. 1983;15:28–33.
• Cohen-Tannoudji J, Einhorn J, Signoret J.P. Ram sexual pheromone: First approach of chemical identification. Physiol. Behav. 1994;56:955–961. [PubMed: 7824597]
• Custer E.E, Berardinelli J.G, Short R.E, Wehrman M, Dair R.A. Postpartum interval to estrus and patterns of LH and progesterone in first-calf suckled beef cows exposed to mature bulls. J. Anim. Sci. 1990;68:1370–1377. [PubMed: 2365649]
• Dagg A.I, Taub A. Flehmen. Mammalia. 1970;34:686–695.
• Danell B, Gopakumar N, Nair M.C.S, Rajagopalan K. Heat symptoms and detection in Surti buffalo heifers. Ind. J. Anim. Res. 1984;5:1–7.
• DeJarnette J.M, Nebel R.L, Marshall C.E. Evaluating the success of sex-sorted semen in US dairy herds from on farm records. Theriogenology. 2009;71:49–51. [PubMed: 18996579]
• Delgadillo J.A, Flores J.A, Veliz F.G, Duarte G, Vielma J, Hernandez H, Fernandez I.G. Importance of the signals provided by the buck for the success of the male effect in goats. Reprod. Nutr. Dev. 2006;46:391–400. [PubMed: 16824448]
• Denhard M, Claus R. Reliability criteria of a bioassay using rats trained to detect estrus-specific odor in cow urine. Theriogenology. 1988;30:1127–1138. [PubMed: 17087902]
• Denhard M, Claus R. Attempts to purify and characterize the estrus-signalling pheromone from cow urine. Theriogenology. 1996;46:13–22.
• Denhard M, Claus R, Pfeiler S, Schopper D. Variation in estrus-related odours in the cow and its dependency on the ovary. Theriogenology. 1991;35:645–652. [PubMed: 16726933]
• Dobson H, Kamonpatana M. A review of female cattle reproduction with special reference to comparison between buffaloes, cows and Zebu. J. Reprod. Fertil. 1986;77:1–36. [PubMed: 3522889]
• Dominic C.J. Chemical communication in animals. J. Sci. Res. (Banaras Hindu University). 1991;41:157–169.
• Dorries K, Regan M, Bruce E.A, Halpern P. Sex difference in olfactory sensitivity to the boar chemosignal, androstenone, in the domestic pig. Anim. Behav. 1991;42:403–411.
• Doving K.B, Trotier D. Structure and function of the vomeronasal organ. J. Exp. Biol. 1998;201:2913–2925. [PubMed: 9866877]
• Dulac C, Wagner S. Genetic analysis of brain circuits underlying pheromone signaling. Annu. Rev. Genet. 2006;40:449–467. [PubMed: 16953793]
• Dzięcioł M, Stańczyk E, Nowak A.N, Niżański W, Ochota M, Kozdrowskia R. Influence of bitches sex pheromones on the heart rate and other chosen parameters of blood flow in stud dogs (Canis familiaris). Res. Vet. Sci. 2012;93:1241–1247. [PubMed: 22401977]
• Estes R. The role of vomeronasal organ in mammalian reproduction. Mammalogy. 1972;36:315–342.
• Fernandez D, Berardinelli J.G, Short R.E, Adair R. The time required for the presence of bulls to alter the interval from parturition to resumption of ovarian activity and reproductive performance in first calf-suckled beef cows. Theriogenology. 1993;39:411–419. [PubMed: 16727221]
• Fernandez D.L, Berardinelli J.G, Short R.E, Adair R. Acute and chronic changes in luteinizing hormone secretion and postpartum interval to estrus in first-calf suckled beef cows exposed continuously or intermittently to mature bulls. J. Anim. Sci. 1996;74:1098–1103. [PubMed: 8726743]
• Fiol C, Ungerfeld R. Biostimulation in cattle: Stimulation pathways and mechanisms of response. Trop. Subtrop. Agroecosyst. 2012;15:1.
• Gaultier E, Bonnafous L, Bought L, Lafont C, Pageat P. Comparison of the efficacy of a synthetic dog-appeasing pheromone with clomiraine for the treatment of separation-related disorders in dogs. Vet. Rec. 2005;156:533–538. [PubMed: 15849342]
• Gelez H, Fabre-Nys C. Role of the olfactory systems and importance of learning in the ewes’ response to rams or their odors. Reprod. Nutr. Dev. 2006;46:401–415. [PubMed: 16824449]
• Gelez H, Archer E, Chesneau D, Campan R, Fabre-Nys C. Importance of learning in the response of ewes to male odor. Chem. Senses. 2004;29:555–563. [PubMed: 15337681]
• Gerall A. An exploratory study of the effect of social isolation variables on the sexual behaviour of male guinea pigs. Anim. Behav. 1963;11:274–282.
• Gokuldas P.P, Yadav M.C, Kumar H, Singh G, Mahmood S, Tomar A.K.S. Resumption of ovarian cyclicity and fertility response in bull exposed postpartum buffaloes. Anim. Reprod. Sci. 2010;121:236–241. [PubMed: 20650574]
• Griffith C.A, Steigerwald E.S, Buffington A.T. Effects of a synthetic facial pheromone on behavior of cats. J. Am. Vet. Med. Assoc. 2000;217:1154–1156. [PubMed: 11043684]
• Hall J.G, Branton C, Stone E.J. Estrus, estrous cycles, ovulation time, time of service and fertility of dairy cows in Louisiana. J. Dairy Sci. 1959;42:1086–1094.
• Halpern M. The organization and function of the vomeronasal system. Ann. Rev. Neuroscience. 1987;10:325–362. [PubMed: 3032065]
• Halpern M, Martínez-Marcos A. Structure and function of the vomeronasal system: An update. Prog. Neurobiol. 2003;70:245–318. [PubMed: 12951145]
• Hawk H.W, Conley H.H, Kiddy C.A. Estrus-related odours in milk detected by trained dogs. J. Dairy Sci. 1984;67:392–397. [PubMed: 6715633]
• Hradecky P. In Czech Literature Review for Graduate Study. University of Veterinary Medicine; Brno, p. Czechoslovakia: 1975. Occurrence of volatile compounds and possibilities of their determination using gas chromatography; p. 185.
• Hradecky P. Volatile fatty acids in urine and vaginal secretions of cows during the reproductive cycle. J. Chem. Ecol. 1986;12:187–196. [PubMed: 24306408]
• Hradecky P, Sis R.F, Klemm W.R. Distribution of flehmen reactions of the bull throughout the bovine estrous cycle. Theriogenology. 1983;20:197–204.
• Ingawale M.V, Dhoble R.L. Buffalo reproduction in India: An overview. Buffalo Bull. 2004;1:4–9.
• Ireland J.J, Murphee R.L, Coulson P.B. Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. J. Dairy Sci. 1980;63:155–160. [PubMed: 7372895]
• Iwata E, Wakabayashi Y, Kakuma Y, Kikusui T, Takeuchi Y, Mori Y. Testosterone-dependent primer pheromone production in the sebaceous gland of male goat. Biol. Reprod. 2000;62:806–810. [PubMed: 10684827]
• Iwata E, Kikusui T, Takeuchi Y, Mori Y. Substances derived from 4-ethyl octanoic acid account for primer pheromone activity for the male effect in goats. J. Vet. Med. Sci. 2003;65:1019–1021. [PubMed: 14532698]
• Izard M.K. Pheromones and reproduction in domestic animals. In: Vandenberg J.G, editor. Pheromones and Reproduction in Mammals. Academic Press; New York: 1983. pp. 253–285. In.
• Izard M.K, Vandenbergh J.G. The effects of bull urine on puberty and calving date in crossbred beef heifers. J. Anim. Sci. 1982;55:1160–1168. [PubMed: 7174555]
• Jacobs V.L, Sir R.F, Coppock C.E. Tongue manipulation of the palate assists estrous detection in the bovine. Theriogenology. 1980;13:353–356. [PubMed: 16725504]
• Karthikeyan K. Tiruchirappalli, India: Bharathidasan University; 2011. Prediction of estrus in murrah buffao species Bubalus bubalis based on volatile compounds from body fluids. Ph.D. thesis.
• Karthikeyan K, Archunan G. J. Buffalo Sci. Vol. 2. 2013. Gas chromatographic mass spectrometric analysis of estrus-specific volatile compounds in buffalo vaginal mucus after initial sexual foreplay; pp. 1–7.
• Karthikeyan K, Muniasamy S, SankarGanesh D, Achiraman S, Ramesh Saravanakumar V, Archunan G. Faecal chemical cues in water buffalo that facilitate estrus detection. Anim. Reprod. Sci. 2013;138:163–167. [PubMed: 23570909]
• Keller M, Meurisse M, Lévy F. Mapping the neural substrates involved in maternal responsiveness and lamb olfactory memory in parturient ewes using Fos imaging. Behav. Neurosci. 2004;118:1274–1284. [PubMed: 15598136]
• Keller M, Meurisse M, Lévy F. Mapping of brain networks involved in consolidation of lamb recognition memory. Neuroscience. 2005;133:359–369. [PubMed: 15885919]
• Kendrick K.M, Lévy F, Keverne E.B. Changes in the sensory processing of olfactory signals induced by birth in sheep. Science. 1992;8:833–836. [PubMed: 1589766]
• Keverne E.B, Lévy F, Guevara-Guzman R, Kendrick K.M. Influence of birth and maternal experience on olfactory bulb neurotransmitter release. Neuroscience. 1993;56:557–565. [PubMed: 7902968]
• Kiddy C.A, Mitchell D.S. Estrus-dated odors in cows: Time of occurrence. J. Dairy Sci. 1984;64:267. [PubMed: 7276312]
• Kiley-Worthington M. The Behavior of Horses. J.A. Allen; London: 1987.
• Kimura R. Volatile substances in feces, of urine and urine-marked feces of feral horses. Can. J. Anim. Sci. 2001;81:411–420.
• Kirkwood R.N, Forbes J.M, Hughes P.E. Influence of boar contact on attainment of puberty in gilts after removal of the olfactory bulbs. J. Reprod. Fertil. 1981;61:193–196. [PubMed: 7452617]
• Klemm W.R, Hawkins G.N, De Los Santos E. Identification of compounds in bovine cervico-vaginal mucus extracts that evoked male sexual behaviour. Chem. Senses. 1987;12:77–87.
• Knight T.W, Lynch P.R. Source of ram pheromones that stimulate ovulation in the ewe. Anim. Reprod. Sci. 1980;3:133–136.
• Knight T.W, Peterson A.J, Payne E. The ovarian and hormonal response of the ewe to stimulation by the ram early in the breeding season. Theriogenology. 1978;10:343–353.
• Krishna N.S, Getchell M.L, Margolis F.L, Getchell V. Differential expression of vomeromodulin and odorant-binding protein, putative pheromone and odorant transporters, in the developing rat nasal chemosensory mucosae. J. Neurosci. Res. 1995;40:54–71. [PubMed: 7714926]
• Ladewig J, Edward O, Price B, Hart L. Flehmen in male goats: Role in sexual behavior. Behav. Neural. Biol. 1980;30:312–332. [PubMed: 7193451]
• Levine E.D, Mills D.S. Long term follow-up of the efficacy of a behavioural treatment programme for dogs with firework fears. Vet. Rec. 2008;162:657–659. [PubMed: 18487586]
• Levy F, Fleming A.S. The neurobiology of maternal behavior in mammals. In: Marshall P.J, Fox N.A, editors. The Development of Social Engagement. Neurobiological Perspectives. Oxford University Press; New York: 2006. pp. 197–246. In.
• Lévy F, Gervais R, Kindermann U, Orgeur P, Piketty V. Importance of beta-noradrenergic receptors in the olfactory bulb of sheep for recognition of lambs. Behav. Neurosci. 1990;104:464–469. [PubMed: 2162183]
• Lévy F, Kendrick K.M, Goode J.A, Guevara-Guzman R, Keverne E.B. Oxytocin and vasopressin release in the olfactory bulb of parturient ewes: Changes with maternal experience and effects on acetylcholine, gamma-aminobutyric acid, glutamate and noradrenaline release. Brain Res. 1995;669:197–206. [PubMed: 7712175]
• Lledo P.M, Alonso M, Grubb M.S. Adult neurogenesis and functional plasticity in neuronal circuits. Nat. Rev. Neurosci. 2006;7:179–193. [PubMed: 16495940]
• Ma W, Klemm W.R. Variations of equine urinary volatile compounds during the oestrous cycle. Vet. Res. Comm. 1997;21:437–446. [PubMed: 9266663]
• Macmillan K.L, Allison A.J, Struthers G.A. Some effects of running bulls with suckling cows or heifers during the premating period. N. Z. J. Exp. Agric. 1979;7:121–124.
• Marinier S.L, Alexander A.J, Wari G.H. Flehmen behaviour in the domestic horse: Discrimination of conspecific odours. Appl. Anim. Behav. Sci. 1988;19:227.
• Martin G.B. Reproductive research on farm animals for Australia—Some long-distance goals. Reprod. Fertil. Dev. 1995;7:967–982. [PubMed: 8848618]
• McGlone J.J, Anderson D.L. Synthetic maternal pheromone stimulates feeding behavior and weight gain in weaned pigs. J. Anim. Sci. 2002;80:3179–3183. [PubMed: 12542158]
• Meurisse M, Chaillou E, Lévy F. Afferent and efferent connections of the cortical and medial nuclei of the amygdala in sheep. J. Chem. Neuroanat. 2009;37:87–97. [PubMed: 18835351]
• Mitchell G, Clark M, Lu R, Caswell J. Localization and functional characterization of pulmonary bovine odorant-binding protein. Vet. Pathol. 2011;48:1054–1060. [PubMed: 20826843]
• Mozūraitis R, Būda V, Kutra J, Borg-Karlson A.-K. p- and m-Cresols emitted from estrous urine are reliable volatile chemical markers of ovulation in mares. Anim. Reprod. Sci. 2012;130:51–56. [PubMed: 22266248]
• Mucignat-Caretta C, Redaelli M, Caretta A. One nose, one brain: Contribution of the main and accessory olfactory system to chemosensation. Front. Neuroanat. 2012;6:1–9. [PMC free article: PMC3494019] [PubMed: 23162438]
• Nordéus K, Båge R, Gustafsson H, Söderquist L. Changes in LH pulsatility profiles in dairy heifers during exposure to oestrous urine and vaginal mucus. Reprod. Domestic. Anim. 2012;47:952–958. [PMC free article: PMC3533760] [PubMed: 22390462]
• Nowak R, Keller M, Levy S. Mother-young relationship in sheep: A model for a multidisciplinary approach of the study of attachment in mammals. J. Neuroendocrinol. 2011;23:1042–1053. [PubMed: 21827554]
• Nowak R, Murphy T.M, Lindsay D.R, Alster P, Anderson R, Berk K. Development of a preferential relationship with the mother by the newborn lamb: Importance of the suckling actiity. Physiol. Behav. 1997;62:681–688. [PubMed: 9284484]
• Pageat P. Pig appeasing pheromones to decrease stress, anxiety and aggressiveness. 2001. US Patent No. 6,169,113, issued January 2, 2001.
• Pageat P, Teyssier Y. Usefulness of a porcine pheromone analogue in the reduction of aggressions between weanlings on penning; behavior study. Congr. Int. Pig Vet. Soc. 1998:413. Birmi UK.
• Paleologou A.M. A study of the cervico-vaginal secretions of cows during the diferent phases of the estrous cycle. J. Inst. Anim. Tech. 1979;30:83–94.
• Patra M.K, Barman P, Kumar H. Potential application of pheromones in reproduction of farm animals–A review. Agri. Rev. 2012;33:82–86.
• Pelosi P. Odorant-binding proteins. Crit. Rev. Biochem. Mol. Biol. 1994;29:199–228. [PubMed: 8070277]
• Poindron P, Levy F, Krehbiel D. Genital, olfactory and endocrine interactions in the development of maternal behavior in the parturient ewe. Psychoneuroendocrinology. 1988;13:99–125. [PubMed: 3287420]
• Poindron O, Otal J, Ferreira G, Keller M, Guesdon V, Nowak R, Levy F. Amniotic fluid is important for the maintenance of maternal responsiveness and the establishment of maternal selectivity in sheep. Animal. 2010;4:2057–2064. [PubMed: 22445380]
• Porter R.H, Levy F, Poindron P, Litterio M, Schaal B, Beyer C. Individual olfactory signatures as major determinants of early maternal discrimination in sheep. Dev. Psychobiol. 1991;24:151–158. [PubMed: 1936579]
• Powers J.B, Winans S.S. Vomeronasal organ: Critical role in mediating sexual behavior of the male hamster. Science. 1975;187:961–963. [PubMed: 1145182]
• Presicce G.A, Brocketta C.C, Chenga T, Foote R.H, Rivard G.F, Klemm W.R. Behavioral responses of bulls kept under artificial breeding conditions to compounds presented for olfaction, taste or with topical nasal application. Appl. Anim. Behav. Sci. 1993;37:273–284.
• Preti G. Method for detecting bovine estrus by determining methyl heptanol concentrations in vaginal secretions. 1984. US Patent No. 4,467,814, issued August 28, 1984.
• Rajanarayanan S. India: Bharathidasan University; 2005. Assessment of flehmen behavior and identification of urinary pheromones in buffaloes (Bubalus bubalis) with special reference to estrus. Ph.D. thesis.
• Rajanarayanan S, Archunan G. Theriogenology. Vol. 61. 2004. Occurrence of flehmen in male buffaloes (Bubalus bubalis) with special reference to estrus; pp. 861–866. [PubMed: 14757472]
• Rajanarayanan S, Archunan G. Identification of urinary sex pheromones in female buffaloes and their influence on bull reproductive behaviour. Res. Vet. Sci. 2011;91:301–305. [PubMed: 21316068]
• Rajkumar R, Karthikeyan K, Archunan G, Huang P.H, Chen Y.W, Ng W.V, Liao C.C. Using mass spectrometry to detect buffalo salivary odorant-binding protein and its post-translational modifications. Rapid Commun. Mass Spectrom. 2010;24:3248–3254. [PubMed: 20972998]
• Rameshkumar K. India: Bharathidasan University; 2000. Chemical characterization of bovine (Bos taurus) urine with special reference to reproductive behavior. Ph.D. thesis.
• Rameshkumar K, Archunan G, Jeyaraman R, Narasimhan S. Chemical characterization of bovine urine with special reference to oestrus. Vet. Res. Commun. 2000;24:445–454. [PubMed: 11085464]
• Rameshkumar K, Achiraman S, Karthikeyan K, Archunan G. Ability of mice to detect estrous odor in bovine urine: Roles of hormones and behavior in odor discrimination. Zool. Sci. 2008;25:349–354. [PubMed: 18459816]
• Ramoni R, Vincent F, Ashcroft A.E, Accornero P, Grolli S, Valencia C, Tegoni M, Cambillau C. Control of domain swapping in bovine odorant-binding protein. Biochem. J. 2002;365:739–748. [PMC free article: PMC1222703] [PubMed: 11931632]
• Rasmussen L.E.L, Anthony J, Martin H, Hess D.L. Chemical profiles of African elephants, Loxodonta africana: Physiological and ecological implications. J. Mammal. 1996;77:422–439.
• Reinhardt V. Flehmen, mounting and copulation among members of a semi-wild cattle herd. Anim. Behav. 1983;31:641–650.
• Rekwot P.I, Ogwu D, Oyedipe E.O, Sekoni V.O. The role of pheromones and biostimulation in animal reproduction. Anim. Reprod. Sci. 2001;65:157–170. [PubMed: 11267796]
• Riley R, Grogan E, McDonnell S. Philadelphia: University of Pennsylvania; 2002. Evaluation of usefulness if equine appeasing pheromone in gentling of foals and yearlings. Thesis.
• Rivard G, Klemm W.R. Chem. Senses. Vol. 14. 1989. Two body fluids containing bovine estrous pheromone(s) pp. 273–279.
• Rivas-Muñoz R, Fitz-Rodríguez G, Poindron P, Malpaux B, Delgadillo J.A. Stimulation of estrous behavior in grazing female goats by continuous or discontinuous exposure to males. J. Anim. Sci. 2007;85:1257–1263. [PubMed: 17085732]
• Roberson M.S, Ansotegui R.P, Berardinelli J.G, Whitman R.W, McInverny M.J. Influence of mature bulls on occurrence of puberty in beef heifers. J. Anim. Sci. 1987;64:1601–1605. [PubMed: 3597174]
• Roberson M.S, Wolfe M.W, Stumpf T.T, Werth L.A, Cupp A.S, Kojima N.D, Wolfe P.L, Kittok R.J, Kinder J.E. Influence of growth rate and exposure to bulls on age at puberty in beef heifers. J. Anim. Sci. 1991;69:292–298. [PubMed: 2066319]
• Salazar I, Quinteiro P.S, Cifuentes J.M. Comparative anatomy of the vomeronasal cartilage in mammals: Mink, cat, dog, pig, cow and horse. Ann. Anat. 1995;177:475–481. [PubMed: 7645743]
• Sambraus H.H, Waring Z. Effect of urine from estrous cows on libido in bulls. Zoologie Saugettierkunde. 1975;40:49–54.
• Sankar R, Archunan G. Flehmen response in bull: Role of vaginal mucus and other body fluids of bovine with special reference to estrus. Behav. Process. 2004;67:81–86. [PubMed: 15182928]
• Sankar R, Archunan G. Discrimination of bovine estrus-related odors by mice. J. Ethol. 2005;23:147–151.
• Sankar R, Archunan G. Identification of putative pheromones in bovine (Bos taurus) feces in relation to estrus detection. Anim. Reprod. Sci. 2008;103:149–153. [PubMed: 17507187]
• Sankar R, Archunan G. Gas chromatographic/mass spectrometric analysis of volatile metabolites in bovine vaginal fluid and assessment of their bioactivity. Int. J. Anal. Chem. 20112011. Article ID 256106. [PMC free article: PMC3202105] [PubMed: 22114602]
• Sankar R, Archunan G. Seasonal effects on estrus behaviours in dairy cattle. Int. J. Adv. Vet. Sci. Tech. 2012;1:28–34.
• Sankar R, Archunan G, Habara Y. Detection of oestrous-related odour in bovine (Bos taurus) saliva: Bioassay of identified compounds. Animal. 2007;1:1321–1327. [PubMed: 22444887]
• Sasada H, Sugiyama T, Yamashita K, Masaki J. Identification of specific odor components in mature male goat during the breeding season. Jpn. J. Zootech. Sci. 1983;54:401–408.
• Scaloni A, Paolini S, Brandazza A, Fantacci M, Bottiglieri C, Marchese S, Navarrini A, Fini C, Ferrara L, Pelosi P. Purification, cloning and characterisation of odorant-binding proteins from pig nasal epithelium. Cell. Mol. Life Sci. 2001;58:823–834. [PubMed: 11437241]
• Schams D, Schallenberger E, Hoffmann B, Karg H. The oestrous cycle of the cow: Hormonal parameters and time relationships concerning oestrus, ovulation, and electrical resistance of the vaginal mucus. Acta Endocrinol. 1977;86:180–192. [PubMed: 578608]
• Schinckel P.G. The effect of the presence of the ram on the ovarian activity of the ewe. Aus. J. Agri. Res. 1954;5:465–469.
• Shelton M. Reproduction and breeding of goats. J. Dairy Sci. 1978;61:994–1010. [PubMed: 29915]
• Short R.E, Bellows R.A, Staigmiller R.E, Berardinelli J.G, Custer E.E. Physiological mechanisms controlling anestrus and infertility in postpartum beef cattle. J. Anim. Sci. 1990;68:799–816. [PubMed: 2180877]
• Signoret J.P. Reproductive behavior of pigs. J. Reprod. Fertil. 1970;11:105–117. [PubMed: 5266385]
• Soto Belloso E, Portillo G, Ramírez L, Soto G, Rojas N, Cruz-Arambulo R. Efecto del destete por noventiséis horas sobre la inducción del celo y fertilidad en vacas mestizas acíclicas. Arch. Latinoam. Anim. Prod. 1997;5:359–361.
• Stahlbaum C.C, Houpt K.A. The role of the flehmen response in the behavioral repertoire of the stallion. Physiol. Behav. 1989;45:1207–1214. [PubMed: 2813545]
• Sugiyama T, Sasada H, Masaki J, Yamashita K. Unusal fatty acids with specific odour from mature male goat. Agri. Biol. Chem. 1981;45:2655–2658.
• Tilbrook A.J, Cameron A.W.N. Ram mating preferences for wooly rather than recently shorn ewes. Appl. Anim. Behav. Sci. 1989;24:301–312.
• Tirindelli R, Dibattista M, Pifferi S, Menini A. From pheromones to behavior. Physiol. Rev. 2009;80:921–956. [PubMed: 19584317]
• Tirindelli R, Mucignat-Caretta C, Ryba N.J.R. Molecular aspects of pheromonal communications in the vomeronasal organ of mammals. Trends Neurosci. 1998;21:482–486. [PubMed: 9829690]
• Underwood E.J, Shier F.L, Davenport N. Studies in sheep husbandry. The breeding season in Merino crossbreeds and British breeds ewes in the agricultural districts. J. Agri. 1944;11:135–143.
• Ungerfeld R. Socio-sexual signaling and gonadal function: Opportunities for reproductive management in domestic ruminants. In: Juengel J.I, Murray J.F, Smith M.F, editors. Reproduction in Domestic Ruminants VI. Nottingham University Press; Nottingham: 2007. pp. 207–221. In. [PubMed: 17491149]
• Ungerfeld R, Silva L. The presence of normal vaginal flora is necessary for normal sexual attractiveness of estrous ewes. Appl. Behav. Sci. 2004;93:245–250.
• Ungerfeld R, Dago A.L, Rubianes E, Forsberg M. Response of anestrous ewes to the ram effect after follicular wave synchronization with a single dose of estradiol-17 beta. Reprod. Nutr. Dev. 2004;44:89–98. [PubMed: 15189013]
• Van den Hurk R. Zeist: Pheromone Information Centre; Brugakker, Netherlands: 2007. Intraspecific chemical communication in vertebrates with special attention to its role in reproduction.
• Vandenbergh J.G. In Encyclopedia of Reproduction. Academic Press; London: 1999. Pheromones in mammals; pp. 764–769.
• Vincent F.I, Ramoni R, Spinelli S, Grolli S, Tegoni M, Cambillau C. Crystal structures of bovine odorant-binding protein in complex with odorant molecules. Eur. J. Biochem. 2004;271:3832–3842. [PubMed: 15373829]
• Weidong M.A, Clement B.A, Klemm W.R. Volatile compounds of bovine milk as related to the stage of the estrous cycle. J. Dairy Sci. 1997;80:3227–3233. [PubMed: 9436103]
• Weigerinck W, Setkus A, Buda V, Borg-Karlson A.K, Mozuraitis R, deGee A. BOVINOSE: Pheromone based sensor system for detecting estrus in dairy cows. Procedia Comput. Sci. 2011;7:340–342.
• Wierzbowski S, Hafez E.S.E. Analysis of copulatory reflexes in the stallion. Proc. Fourth Inter. Cong. Anim. Reprod. The Hague. 1961;2:176–179.
• Williamson N.B, Morris R.S, Blood D.C, Cannon C.M. A study of oestrus behaviour and oestrus detection methods in a large commercial dairy herd. Vet. Rec. 1972;91:50–58. [PubMed: 5073596]
• Winans S, Powers J.B. Olfactory and vomeronasal deafferentation of male hamsters: Histological and behavioral analyses. Brain Res. 1977;126:325–344. [PubMed: 861723]
• Wöhrmann-Repenning A. Comparative anatomical studies of the vomeronasal complex and the rostral palate of various mammals. I. Gegenbaurs Morphologisches Jahrbuch. 1984;130:501–530. [PubMed: 6489726]
• Wright I.A.S, Rhind M, Whyte T.K, Smith A.J. Effect of body condition at calving and feeding level after calving on LH profiles and duration of the post-partum anoestrous period in beef cows. Anim. Prod. 1992;55:41–46.
• Ziegler T.E, Epple G, Snowdon C.T, Porter T.A, Belcher A.M, Kuderling I. Detection of the chemical signals of ovulation in the cotton-toptamarin. Saguinus oedipus. Anim. Behav. 1993;45:313–322.